The present invention relates to improved continuous (immortalized) cell lines derived from normal human skin tissues, in particular keratinocytes and melanocytes which retain the ability to express differentiation proteins characteristic of differentiated melanocytes or keratinocytes, even in high passages. The present invention also relates to novel serum free media which do not require the use of feeder cells.
The production of immortalized cell lines derived from human skin tissues has been previously described. In general such methods comprise transfection or transformation of human skin cells, e.g., keratinocytes and melanocytes, cultured in vitro with agents which provide for immortalization.
Immortalization refers to the production of cells which are able to be cultured for prolonged time periods in vitro, ideally indefinitely. These cells are also referred to as continuous cell lines. By contrast non-immortalized cells are only capable of growing for a finite number of cell divisions in vitro. Immortalized cells are highly desirable because they provide a stable, potentially infinite supply of cells having defined characteristics. Conventional agents for producing immortalized cell lines and immortalized human skin cell lines in particular include, e.g., viruses, recombinant viruses, and plasmids which contain DNA sequences which provide for immortalization.
Probably the most common method of producing immortalized human cell lines involves the use of SV40 sequences and more specifically the SV40 large T antigen DNA as an immortalizing agent. For example, Steinberg et al., J. Cell Phys., 123:117-125 (1985); Reddel et al., U.S. Pat. No.4,885,238 issued on Dec. 5, 1989; Major, U.S. Pat. No. 4,707,448 issued on Nov. 17, 1987; Stoner etal., Cancer Res., 51:365-371 (1991); Chopra et al., In Vitro Cell Dev. Biol., 30A:539-546 (1994); Chopra et al., In Vitro Cell Dev. Biol., 27A:763-765 (1991); Christian etal., Cancer Res., 47:6066-6073 (1987); Rhim et al., Science, 227:1250-1252 (1985); and Grubman et al., Gastrointest. Liver Physiol., 29:G1060-1070 (1994) teach the use of SV40 vectors and SV40 large T antigen sequence containing vectors to produce immortalized human cell lines. The introduction of such sequences is generally effected by infection using SV40 virus or with a hybrid adenovirus-12 SV40 hybrid virus or by transfection of cells with a recombinant plasmid containing the Rous sarcoma virus long terminal repeat and the Ori-SV40 early region by strontium phosphate co-precipitation. (See Brash et al., Mol. Cell Biol., 7:2031-2034, (1987)).
Another known method for producing immortalized cell lines, and immortalized human keratinocytes in particular, involves transfection or infection of cells with human papillomavirus DNA sequences. For example, U.S. Pat. No. 5,376,542 by Schlegel issued on Dec. 27, 1994 describes immortalization of human epithelial cells with isolated HPV-16, 18, 31, 33 or 35 E6 and E7 genes or the E7 gene alone to produce non-tumorigenic immortalized cell lines. Also, Barbosa et al., Oncogene, 4:1529-1532 (1989); and Muinger et al., J. Virol., 63(10):4417-4421 (1989) teach the use of HPV-16 and HPV-18 E6 and E7 genes to produce immortalized human keratinocytes.
However, while numerous groups have reported immortalized keratinocyte cell lines, and their usage in in vitro assays, previous immortalized keratinocyte cell lines and melanocyte cell lines have typically exhibited one or more properties which render their usage disadvantageous. For example, previously reported immortalized keratinocytes have exhibited one or more of the following characteristics: (i) reduction or loss of expression of differentiation markers, e.g., proteins which are expressed by normal differentiated keratinocytes, and (ii) altered growth characteristics in tissue culture.
For example, Jetten et al, J. Invest. Dermatol., 92:203-209 (1989) reports SV40 immortalized keratinocytes obtained after high passage number ( greater than passage 12) using the vector NHEK-SV40-T8-1 which are unable to differentiate. Similarly, Bernard et al, Cancer Res., 45:1707-1716 (1985) report isolation of an immortalized keratinocyte cell line referred to as SVK14 which is reported to be almost completely unable to differentiate. Also, this cell line shows no expression of keratins K1/10 ( greater than 53 kD) and the 50 kD keratin (keratin K14), which proteins are normally expressed by differentiated keratinocytes.
Still further, Steinberg et al, J. Cell. Physiol., 123:117-125 (1985) report SV40 transformed keratinocytes which gradually lose the ability to express keratins which are characteristic of the normal keratin cytoskeleton. This loss of normal keratin expression occurs after about 10-15 passages. Also, Hronis et al, Cancer Res., 44:5797-5804 (1984) teach SV40 DNA immortalized keratinocytes which have lost the ability to produce K5, K6, K14/15, K16 and K17 keratins and involucrin which proteins are characteristic of normal differential keratinocytes. Still further, Morris et al, Proc. Natl. Acad. Sci. USA, 82:8498-8502 (1985) teach SV40 immortalized keratinocytes which at higher passages ( greater than passage 14) exhibit highly reduced expression of Class II and Class I keratins. For example, these keratinocytes exhibit almost no expression of K13 (ILd).
Also, Banks-Schlegel et al, J. Cell. Biol., 96:330-337 (1983) disclose SV40 immortalized keratinocytes which exhibit altered growth characteristics in tissue culture. For example, unlike normal keratinocytes, these immortalized cells require a 3T3 feeder layer to grow.
Previously described methods for producing immortalized human keratinocytes and melanocytes have typically used the feeder cell technique (wherein fibroblasts usually function as the xe2x80x9cfeederxe2x80x9d cells) and generally. culture cells in serum-containing medium. For example, Sexton et al., xe2x80x9cStable transfection of human keratinocytes: HPV immortalization,xe2x80x9d Keratinocyte Methods, eds., Leigh, I. M. et al., University Press, 179-180, (1994); Garlick, xe2x80x9cRetroviral Vectors,xe2x80x9d Keratinocyte methods, (eds. Leigh I. M. et al., Cambridge University Press, 181-183 (1994)) teach the use of fetal calf serum containing medium and feeder cells in the isolation and production of immortalized keratinocytes.
The use of serum-free medium during the isolation and production of immortalized epithelial cells, and specifically human keratinocytes has been previously described. For example, Barbosa et al., Oncogene, 4:1529-1532 (1989) describe initially culturing human keratinocytes transfected by electroporation or lipofection in low calcium, serum-free medium until confluence.
However, notwithstanding what has been previously reported, there still exists a significant need in the art for immortalized human keratinocytes and melanocytes which possess improved properties, in particular which maintain the differentiation potential of normal keratinocytes and melanocytes and which express differentiation proteins characteristic of differentiated melanocytes and keratinocytes, even after high passages. Such cells would be highly beneficial for many usages, in particular in assays which require differentiated skin cells. There further exists a need in the art for improved culture media capable of maintaining primary and immortalized keratinocytes and melanocytes, as well as improved culturing methods which do not require the use of feeder cells.
Towards that end, it is an object of the invention to produce improved continuous (immortalized) cell lines derived from normal human skin tissue, especially immortalized keratinocytes and/or melanocyte cell lines derived from normal human skin tissue which maintain the ability to differentiate and to express differentiation proteins even after high passages. More specifically, it is an object of the invention to obtain immortalized keratinocytes which maintain the ability to express keratins, cytochromes, as well as other differentiation proteins, e.g., involucrin, filaggrin and loricrin, which are either not expressed or poorly expressed by conventional immortalized keratinocyte cell lines. It is still another object of the invention to obtain immortalized keratinocytes and melanocytes which express enzymes which are normally expressed by differentiated keratinocytes and melanocytes, especially phase II enzymes such as glutathione-S-transferases as well as enzymes and/or proteins which are involved in cellular oxidation and inflammatory responses.
It is another object of the invention to provide novel serum free media for culturing, producing and maintaining normal or continuous keratinocytes and/or melanocytes in tissue culture. These novel serum free media are also useful for isolating, establishing and immortalizing human skin cells for obtaining continuous melanocytes and keratinocytes according to the invention. It is thus a specific object of the invention to provide a fully defined culture medium (without unknown or ill-defined supplements) for culturing keratinocytes without feeder cells containing epinephrine which has surprisingly been discovered to be a strong growth potentiator of normal keratinocytes in serum-free medium.
It is another object of the invention to provide a novel method for isolating, establishing and immortalizing human skin cells for obtaining continuous melanocyte and keratinocyte cell lines derived from normal skin tissue, said method using serum-free media, notably those according to the invention, and a cell attachment cocktail containing fibronectin, BSA and type I collagen without xe2x80x9cfeeder cellsxe2x80x9d (e.g., fibroblasts).
It is another object of the invention to provide primary keratinocyte or melanocytes produced under serum free conditions without the use of any feeder cells, said primary keratinocytes and melanocytes being used for skin grafting and in ex vivo genetic therapy.
It is another object of the invention to provide methods of using such novel and improved continuous keratinocyte and melanocyte cell lines, e.g., for immunological, pharmacological, photo- and chemotoxicological skin reaction assays and for the expression of heterologous genes.